Hamid Ashrafi

Blackberries (Rubus spp.) are the fourth most economically important berry crop worldwide. Genome assemblies and annotations have been developed for Rubus species in subgenus Idaeobatus, including black raspberry (R. occidentalis), red raspberry (R. idaeus), and R. chingii, but very few genomic resources exist for blackberries and their relatives in subgenus Rubus. Here we present a chromosome-length assembly and annotation of the diploid blackberry germplasm accession “Hillquist” (R. argutus). “Hillquist” is the only known source of primocane-fruiting (annual-fruiting) in tetraploid fresh-market blackberry breeding programs and is represented in the pedigree of many important cultivars worldwide. The “Hillquist” assembly, generated using Pacific Biosciences long reads scaffolded with high-throughput chromosome conformation capture sequencing, consisted of 298 Mb, of which 270 Mb (90%) was placed on 7 chromosome-length scaffolds with an average length of 38.6 Mb. Approximately 52.8%...

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops’ relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the developme...

The feeding habits of adult Brachycera are understudied and may provide important context for understanding invasive pest biology, as with the polyphagous small fruit pest Drosophila suzukii. We developed molecular methods to study adult D. suzukii gut content in order to understand its feeding habits. We designed and verified two primer pairs specific for either blueberries or blackberries and used a qPCR melt curve analysis to determine whether we can detect the presence or absence of berry feeding by adult flies. In a laboratory assay, the blueberry fly meal DNA can be detected for longer periods than the blackberry meal DNA. Generally, female gut contents are less variable than male gut contents. We also tested recently emerged flies that were not fed as adults but developed as larvae in either blueberries or blackberries. Some adult flies from each fruit had detectable fruit DNA in their gut, which could be due to pupal meconium feeding after emergence. Next, we aimed to test t...

Vavilov (1951) placed the center of origin of cultivated carrot in Central Asia, and an analysis of molecular diversity in wild and cultivated carrots from around the world demonstrated that wild carrots from Central Asia were more similar to cultivated carrots (Iorizzo et al., 2013), confirming Vavilov’s conclusions. Carrots may have been cultivated as a root crop in the Roman Empire, with extensive cultivation first recorded around 900 AD in Central Asia – Afghanistan in particular (Stolarczyk and Janick, 2011; Banga, 1963). Color has played an important role in the history of carrot domestication. The first Central Asian carrots were yellow or purple, and in the early 1500s, orange carrots were noted in still life paintings and some written accounts in Europe. Central Asian carrots spread first to the west beginning in the 900s, through the Middle East, North Africa, and then Europe; and to the east to South and North Asia (Banga, 1963). Orange carrots are grown globally today but yellow, purple, red, and white carrot land races, and some modern cultivars, are grown on a more limited scale in several parts of the world.Fil: Simon, Phillip. University of Wisconsin; Estados UnidosFil: Iorizzo, Massimo. University of Wisconsin; Estados UnidosFil: Ellison, Shelby. University of Wisconsin; Estados UnidosFil: Senalik, Douglas A.. University of Wisconsin; Estados UnidosFil: Zeng, Peng. Beijing Genome Institute; ChinaFil: Pimchanok, Satapoomin. Kasetsart University; TailandiaFil: Huang, Jaiying. Beijing Genome Institute; ChinaFil: Bowman, Megan. Van Andel Research Institute; Estados UnidosFil: Iovene, Marina. National Research Council; ItaliaFil: Sanseverino, Walter. Sequentia Biotech; EspañaFil: Cavagnaro, Pablo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Departamento de Producción Agropecuaria. Cátedra de Horticultura y Floricultura; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria La Consulta; ArgentinaFil: Yildiz, Mehtap. Yuzuncu Yin University. Faculty Of Agriculture; TurquíaFil: Macko-Podgorni, Alicja. University Of Agriculture In Krakow; PoloniaFil: Moranska, Emilia. University Of Agriculture In Krakow; PoloniaFil: Grzebelus, Ewa. University Of Agriculture In Krakow; PoloniaFil: Grzebelus, Dariusz. University Of Agriculture In Krakow; PoloniaFil: Ashrafi, Hamid. University of North Carolina; Estados UnidosFil: Zheng, Zhijun. Beigin Genome Institute; ChinaFil: Cheng, Shifeng. Beigin Genome Institute; ChinaFil: Spooner, David. University of Wisconsin; Estados UnidosFil: Van Deynze, Allen. University of California at Davis; Estados Unido

Genotyping‐by‐sequencing (GBS) was employed to construct a highly saturated genetic linkage map of a tomato (Solanum lycopersicum L.) recombinant inbred line (RIL) population, derived from a cross between cultivar NC EBR‐1 and the wild tomato S. pimpinellifolium L. accession LA2093. A pipeline was developed to convert single nucleotide polymorphism (SNP) data into genomic bins, which could be used for fine mapping of quantitative trait loci (QTL) and identification of candidate genes. The pipeline, implemented in a python script named SNPbinner, adopts a hidden Markov model approach for calculation of recombination breakpoints followed by genomic bins construction. The total length of the newly developed high‐resolution genetic map was 1.2‐fold larger than previously estimated based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)–based markers. The map was used to verify and refine QTL previously identified for two fruit quality traits in the R...

Mummy berry disease, caused by the fungal pathogen <i>Monilinia vaccinii-corymbosi</i> (<i>Mvc</i>), is one of the most economically important diseases of blueberries in North America. <i>Mvc</i> is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of <i>Mvc</i> and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for <i>Mvc</i>. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. Macrosynteny analysis revealed syntenic regions between the <i>Mvc </i>genome and genomes of taxonomically related fungi. A total of 9,405 gene model...

Background Tripidium ravennae is a cold-hardy, diploid species in the sugarcane complex (Poaceae subtribe Saccharinae) with considerable potential as a genetic resource for developing improved bioenergy and ornamental grasses. An improved understanding of the genetic regulation of reproductive processes (e.g., floral induction, inflorescence development, and seed development) will enable future applications of precision breeding and gene editing of floral and seed development. In particular, the ability to silence reproductive processes would allow for developing seedless forms of valuable but potentially invasive plants. The objective of this research was to characterize the gene expression environment of reproductive development in T. ravennae. Results During the early phases of inflorescence development, multiple key canonical floral integrators and pathways were identified. Annotations of type II subfamily of MADS-box transcription factors, in particular, were over-represented i...

Mummy berry disease, caused by the fungal pathogen Monilinia vaccinii-corymbosi (Mvc), is one of the most economically important diseases of blueberries in North America. Mvc is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of Mvc and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for Mvc. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. A total of 9399 gene models were predicted and annotated, and BUSCO analysis revealed that 98% of 1,438 searched conserved eukaryotic genes were present in the predicted gene set. Potential effectors were identified, and the mating-type (MAT) locus was cha...

A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry (Rubus subgenus Rubus). Three regions conserved among apple (Malus × domestica), peach (Prunus persica), and alpine strawberry (Fragaria vesca) were identified that contained previously detected sweetness-related quantitative trait loci (QTLs) in at least two of the crops. Sugar related genes from these conserved regions and 789 sugar-associated apple genes were used to identify 279 Rubus candidate transcripts. A Hyb-Seq approach was used in conjunction with PacBio sequencing to generate haplotype level sequence information of sugar-related genes for 40 cultivars with high and low soluble solids content from the University of Arkansas and USDA blackberry breeding programs. Polymorphisms were identified relative to the 'Hillquist' blackberry (R. argutus) and ORUS 4115-3 black raspberry (R. occidentalis) genomes and tested for their association with soluble so...

Most cultivars of tomato (Lycopersiconesculentum) are susceptible to early blight (EB), a devastating fungal (Alternariasolani) disease of tomato in the northeast and eastern part of the U.S. The most economic and durable measure of disease control is by using genetic resistance. There is limited EB resistance within the cultivated tomato. However, genetic sources of resistance exist within the tomato-related wild species L. hirsutum and L. pimpinellifolium. Early blight resistance does not follow the gene-for-gene model of host–pathogen interaction. Mapping QTLs conferring horizontal resistance is an effective approach for studying complex resistance traits such as EB. We have developed F2, F3, F4, and an F2:7 derived RIL population of a L. esculentum× L. pimpinellifolium cross and evaluated them for EB resistance under field conditions. Genetic maps were constructed based on the F2 (including 256 RFLP, EST and RGA markers) as well as the RIL population (including over 220 RFLP and...

Early blight (EB), caused by the fungus Alternaria solani, is a destructive disease of tomato (Lycopersicon esculentum) worldwide. Sources of genetic resistance have been identified within related wild species, including green-fruited L. hirsutum and red-fruited L. pimpinellifolium. We have employed traditional protocols of plant breeding and contemporary molecular markers technology to discern the genetic basis of EB resistance and develop tomatoes with improved resistance. Backcross breeding has resulted in the development of germplasm with improved resistance; however, linkage drag has been a major obstacle when using L. hirsutum as a donor parent. To identify and map QTLs for EB resistance, we used several filial and backcross populations derived from interspecific crosses between L. esculentum and either L. hirsutum or L. pimpinellifolium. In each population, an average of seven resistance QTLs were detected. While similar QTLs were detected in different generations of the same...

Fruits, as an important part of the human diet, have been under strong selection during domestication. In general, continued directed selection has led to varieties having larger fruit with greater shape variation and tremendous increases in fruit mass. Common cultivated peppers ( L.) are found in a wide range of sizes and shapes. Analysis of genetic relatedness and population structure has shown that the large-fruited, nonpungent types have reduced diversity and comprise a highly structured group. To explore this population structure, a statistical method for detecting fixation within subpopulations was applied to a set of 21 pungent and 19 nonpungent lines that represent the pepper breeding germplasm. We have identified 17 blocks within the pepper genome that are conserved among nonpungent large-fruited varieties. To determine if these regions were fixed by selection on fruit size or pungency, quantitative trait loci (QTLs) from seven studies along with capsaicin biosynthesis gene...

The Capsicum genus (Pepper) is a part of the Solanacae family. It has been important in many cultures worldwide for its key nutritional components and uses as spices, medicines, ornamentals and vegetables. Worldwide population growth is associated with demand for more nutritionally valuable vegetables while contending with decreasing resources and available land. These conditions require increased efficiency in pepper breeding to deal with these imminent challenges. Through resequencing of inbred lines we have completed a valuable haplotype map (HapMap) for the pepper genome based on single-nucleotide polymorphisms (SNP). The identified SNPs were annotated and classified based on their gene annotation in the pepper draft genome sequence and phenotype of the sequenced inbred lines. A selection of one marker per gene model was utilized to create the PepperSNP16K array, which simultaneously genotyped 16 405 SNPs, of which 90.7% were found to be informative. A set of 84 inbred and hybri...

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem develo...

Modern tomato (Solanum lycopersicum) varieties are bred for uniform ripening (u) light green fruit phenotypes to facilitate harvests of evenly ripened fruit. U encodes a Golden 2-like (GLK) transcription factor, SlGLK2, which determines chlorophyll accumulation and distribution in developing fruit. In tomato, two GLKs—SlGLK1 and SlGLK2—are expressed in leaves, but only SlGLK2 is expressed in fruit. Expressing GLKs increased the chlorophyll content of fruit, whereas SlGLK2 suppression recapitulated the u mutant phenotype. GLK overexpression enhanced fruit photosynthesis gene expression and chloroplast development, leading to elevated carbohydrates and carotenoids in ripe fruit. SlGLK2 influences photosynthesis in developing fruit, contributing to mature fruit characteristics and suggesting that selection of u inadvertently compromised ripe fruit quality in exchange for desirable production traits.

Modern tomato (Solanum lycopersicum) varieties are bred for uniform ripening (u) light green fruit phenotypes to facilitate harvests of evenly ripened fruit. U encodes a Golden 2-like (GLK) transcription factor, SlGLK2, which determines chlorophyll accumulation and distribution in developing fruit. In tomato, two GLKs—SlGLK1 and SlGLK2—are expressed in leaves, but only SlGLK2 is expressed in fruit. Expressing GLKs increased the chlorophyll content of fruit, whereas SlGLK2 suppression recapitulated the u mutant phenotype. GLK ...

Blueberries (Vaccinium spp.) comprise a broad range of perennial woody species. Introgression of native species into cultivated germplasm has adapted Vaccinium germplasm to a range of climates and growing conditions for cultivated blueberry. Genetic differences signify phenotypic variance that is observed among blueberry accessions. In addition, variability in geographic and climatic growing conditions between environments or within the same environment across different years may further affect fruit and plant phenotypic expression. As a result, a phenotype is a function of genetic background (G), environment (E), and their interaction (G × E). In addition, other temporally regulated factors such as year (Y) and harvest time (H) impact plant and fruit quality phenotypic variation. Our research aimed to assess the genotypic performance of five blueberry cultivars, including ‘Echota’, ‘O’Neal’, ‘Reveille’, ‘Summit’, and ‘Sunrise’. The selected cultivars were phenotyped for various fru...

Developing new blueberry cultivars requires plant breeders to be aware of current and emerging needs throughout the supply chain, from producer to consumer. Because breeding perennial crop plants (such as blueberry) is time- and resource-intensive, understanding and targeting priority traits is critical to enhancing the efficiency of breeding programs. This study assesses blueberry industry breeding priorities for fruit and plant quality traits based on a survey conducted at commodity group meetings across nine U.S. states and in British Columbia (Canada) between Nov. 2016 and Mar. 2017. In general, industry responses signaled that the most important trait cluster was fruit quality including the firmness, flavor, and shelf life. Fruit quality traits affect price premiums received by producers; influence consumer’s preferences; and have the potential to increase the feasibility of mechanical harvesting, all critical to the economic viability of the industry. There were differences ac...

The US pepper industry in 2012 was valued at $802.7 M. One fifth of this value is from chile (hot-type) peppers. One of the biggest problems for the pepper industry is Phytophthora capsici (Pc) in which all commercial varieties suffer yield losses despite good management practices and available landraces with high levels of resistance. Moreover, breeding resistance to Pc is complicated by the dynamic array of races of Phytophthora found in fields over time and variable resistance across plant varieties and tissue types. A high density map with 3892 markers was generated in a set of recombinant inbred lines derived from the highly resistant Capsicum annuum accession Criollo de Morelos-334 (CM334) and Early Jalapeño. These lines have been systematically screened for root rot resistance against a set of isolates defined by differential analyses collected from Mexico, New Mexico, California, Michigan and Tennessee. QTL effective across isolates and specific to isolates have been identif...

Early blight (EB), caused by fungus Alternaria solani Sorauer, is a destructive disease of tomato (Lycopersicon esculentum Mill.) in the U.S. and elsewhere. Sources of genetic resistance have been identified within tomato related wild species, in particular green-fruited L. hirsutum Humb. and Bonpl. and red-fruited L. pimpinellifolium (Jusl.) Miller. Resistance was previously reported to be horizontal and quantitative, and transmitted as a dominant, additive, or recessive trait, depending on the cross. Furthermore, EB resistance does not follow the gene-for-gene model of host-pathogen interaction. We have used traditional protocols of plant genetics and breeding and contemporary molecular-marker technology to better understand the genetic basis of EB resistance and to develop tomatoes with improved resistance. Traditional backcross breeding has resulted in development of germplasm with improved resistance, however, linkage drag has been a major obstacle, in particular when using L. ...

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SNP-based genetic analysis, manipulation and improvement of Upland cotton (Gossypium hirsutum) is limited by the lack of large numbers of SNPs. Streamlined large scale genomic analysis is presently limited by the modest numbers of intraspecific SNPs available. SNP development via NGS assemblies from normalized transcriptome was quite successful for wild species, but less so for intraspecific SNPs. Limited intraspecific polymorphism exists between lines and cultivars, and when combined with conservation of exon sequences, the yields of SNPs were relatively low and insufficient in number. In order to determine the applicability of current intraspecific SNPs, identified loci need to be screened against a large number of diverse undomesticated and improved lines. The KASPar assay of Kbiosciences was used to assess a large sample of Gossypium hirsutum lines. Here we report on the results of screening existing intraspecific SNPs on a large population of upland cotton.

Molecular breeding of pepper (Capsicum spp.) has been hampered by the paucity of molecular markers. This is primarily due to lack of availability of the pepper genome sequence and limited available sequence resources. However, during the past few years we have developed a wealth of genetic information for pepper including two ultra-saturated maps of pepper using an Affymetrix pepper GeneChip®. In spite of being useful in genetic map construction and QTL mapping, using the Affymerix GeneChip® is not technically and economically feasible for many small laboratories for marker discovery and breeding. In recent years with the more cost effective sequencing technologies such as Illumina, sequencing of expressed genes (transcriptomes), gene discovery and allele mining is no longer insurmountable. In order to exploit the speed and scale of data from new sequencing technologies and in an effort to enrich the sequence resources of pepper, we sequenced transcriptome sequences (RNAseq) of thre...